Strong impetus exists for developing improved modes of insulin delivery for the treatment of diabetes mellitus. The insertion of appropriately regulated insulin genes into non-islet tissues is a potential strategy for the treatment of type I diabetes, in which islet cells are destroyed by autoimmune mechanism. The objective of the proposed project is to explore an approach to gene therapy for diabetes for engineering glucose regulated insulin production in extra-pancreatic sites. Our approach will be to target insulin expression to hepatocytes and intestinal epithelial cells in vivo using an insulin gene construct driven by the liver-type pyruvate kinase (L-PK) promoter. Since L-PK promoter activity is stimulated by glucose and blocked by glucagon and cyclic AMP, we expect that insulin synthesis and secretion will increase after a carbohydrate meal, and that possible over-production of insulin leading to severe hypoglycemia may be prevented by the cAMP-mediated actions of glucagon and epinephrine. Since the L-PK promoter requires permissive amounts of insulin to be active, a second gene construct expressing insulin from a modified metallothionein promoter will be transferred along with the L-PK/insulin gene to provide a basal level of insulin. Double gene cassettes will be packaged into adeno-associated virus vectors and transferred in vivo to mice rendered diabetic by ablation of pancreatic beta cells using the drug streptozotocin. The time course of L-PK/insulin gene activation and repression will be determine after glucose loads and during insulin-induced hypoglycemia. Possible amelioration of the diabetic state will be assessed by oral glucose tolerance tests and measurement of glycohemoglobin levels. New initiatives in gene therapy will undoubtedly require the development of control systems to achieve the desired expression level for varying physiological or therapeutic circumstances. This project will assess a new therapeutic approach to diabetes and may also serve as a model for future attempts to engineer control systems for gene transfer. Furthermore, the Research Center Award will enhance my development as a physician-scientist and allow me to reach my long-term goal of an independent research career in academic medicine.